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In vitro processing of the human alkyl-dihydroxyacetonephosphate synthase precursor
1Institute for Biomembranes, Utrecht University, Utrecht, The Netherlands.
Archives of Biochemistry and Biophysics
|July 23, 1999
Summary
A peroxisomal enzyme crucial for ether phospholipid synthesis is processed by a cysteine protease. This processing occurs within peroxisomes and does not alter the enzyme's activity.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Alkyl-dihydroxyacetonephosphate synthase (ADFPS) is a peroxisomal enzyme essential for ether phospholipid biosynthesis.
- ADFPS is synthesized with a cleavable N-terminal presequence containing the peroxisomal targeting signal type 2 (PTS2).
Purpose of the Study:
- To investigate the processing of human alkyl-dihydroxyacetonephosphate synthase.
- To characterize the protease responsible for processing ADFPS.
- To determine the effect of processing on ADFPS activity.
Main Methods:
- In vitro synthesis and expression of human ADFPS precursor in E. coli.
- Incubation with guinea pig liver organellar fraction and purified peroxisomes.
- Radiosequencing for protein identification.
- Enzyme activity assays.
- Protease characterization (pH optimum).
Main Results:
- Human ADFPS precursor was processed to a mature form by a guinea pig liver organellar fraction.
- The processing protease was identified as a cysteine protease with a pH optimum of 6.5.
- Exogenously added ADFPS precursor was imported and processed in purified peroxisomes.
- Processing did not affect the enzymatic activity of ADFPS.
Conclusions:
- A specific cysteine protease in peroxisomes processes the human ADFPS precursor.
- The presequence is not essential for ADFPS activity.
- This study elucidates a key step in ether phospholipid biosynthesis regulation.